The present invention relates to methods for the transformation and regeneration of transformed embryogenic tissue of coniferous plants. In particular, the invention relates to improved methods for transforming embryogenic tissue of coniferous plants and for regenerating transformed embryogenic tissue of coniferous plants. The invention is well suited to the transformation and regeneration of transformed embryogenic tissue of plants of the subgenus Pinus of pines.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended Bibliography.
Reforestation, the controlled regeneration of forests, has become an integral part of forest management in order to secure a renewable and sustainable source of raw material for production of paper and other wood-related products. Forest trees can be regenerated by either sexual or asexual propagation. Sexual propagation of seedlings for reforestation has traditionally been the most important means of propagation, especially with coniferous species.
Tree improvement programs with economically important conifers (e.g., Pinus, Picea, and Pseudotsuga species) have applied genetic principles of selection and breeding to achieve genetic gain. Based on the results of progeny tests, superior maternal trees are selected and used in “seed orchards” for mass production of genetically improved seed. The genetic gain in such an open-pollinated sexual propagation strategy is, however, limited by the breeder's inability to control the paternal parent. Further gains can be achieved by control-pollination of the maternal tree with pollen from individual trees whose progeny have also demonstrated superior growth characteristics. Yet sexual propagation results in a “family” of seeds comprised of many different genetic combinations (known as siblings), even though both parents of each sibling seed are the same. As not all genotype combinations are favorable, the potential genetic gain is reduced due to this genetic variation among sibling seeds.
In addition to these genetic limitations, large-scale production of control pollinated seeds is expensive. These economic and biological limitations on large-scale seed production have caused considerable interest to develop in the industry for applying asexual methods to propagate economically important conifers.
The use of asexual propagation permits one to apply what is known as a very high selection intensity (that is, to propagate only progeny showing a very high genetic gain potential). These highly desirable progeny have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to multiply genetically select individuals while avoiding a concomitant reduction of genetic gain due to within-family variation. Asexual propagation of trees can be accomplished by methods of grafting, vegetative propagation, and micropropagation. Micropropagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. In addition, somatic embryogenic cultures can easily be preserved in liquid nitrogen. Having a long-term cryogenic preservation system offers immense advantages over other vegetative propagation systems which attempt to maintain the juvenility of stock plants.
One source of new genetic material for use in reforestation or tree improvement programs is plant tissue that has been transformed to contain one or more genes of interest. Genetic modification techniques enable one to insert exogenous nucleotide sequences into an organism's genome. A number of methods have been described for the genetic modification of plants, including transformation via biolistics and Agrobacterium tumefaciens. All of these methods are based on introducing a foreign DNA into the plant cell, isolation of those cells containing the foreign DNA integrated into the genome, followed by subsequent regeneration of a whole plant.
A significant problem in production of transgenic plants is how to recover only transformed cells following transformation, while causing minimal perturbations to their health so that they can proliferate, give rise to differentiating cultures and ultimately regenerate transgenic plants.
It is well known that embryogenic cultures, in general, and pine embryogenic cultures, specifically, can experience significant decline in regeneration potential under stressful culture conditions. Stresses to the cells during and after transformation can include the perturbations of the transformation process (which may include co-cultivation with Agrobacteria, bombardment with microprojectiles, chemical treatments, electroporation or mechanical shearing), any measures that allow preferential growth of transformed cells while selectively killing or depressing the growth or regeneration of untransformed cells (referred to as “selection”), exudates released from dying cells in the culture, and/or the elicitation of transgene activity in the transformed cells (for “positive selection” or detection of the activity of “visual marker genes”). It stands to reason that when transformed cells are not maintained in sufficient health to allow their survival through these stresses, not only will they fail to give rise to transgenic plants, they may never be detected as transformed in the first place.
In a plant genetic transformation process using Agrobacterium tumefaciens as the transforming agent, a usual step is to place the infected plant tissue, after a suitable “co-cultivation period”, into a liquid medium or onto the surface of a gelled medium which incorporates an eradicant for the Agrobacterium. This is done to kill the Agrobacterium, which, after it has accomplished gene transfer into the plant, is a hazard to sterile culture and subsequent good growth of the plant material. Eradication usually involves multiple transfers of the plant cells into uncontaminated media containing antibiotics such as ticarcillin, carbenicillin, or a cephalosporin. The antibiotics are normally incorporated into every stage of the medium following transformation, to prevent Agrobacterium contamination from resurging.
Regeneration of transformed plants from transformed cultures of pine has been difficult. Reports of pine transformation and regeneration include the following:
U.S. Pat. No. 4,459,355 (Cello and Olsen, 1984) describes a method for using Agrobacterium tumefaciens to transform plant cells. The patent claims transformation of any dicotyledon or any gymnosperm (e.g. loblolly pine, cedar, Douglas fir). However, no example of transformation of any gymnosperm is given. Thus, a claim of stable transformation of pines following inoculation with Agrobacterium tumefaciens was allowed in U.S. Pat. No. 4,886,937 (Sederoff et al., 1989).
U.S. Pat. No. 4,886,937 also claims the transformed pine obtained from inoculation with Agrobacterium tumefaciens. However, no transformed pine plants were obtained in the examples, which are restricted to formation of non-regenerable galls following inoculation of seedlings. Further work by researchers in the same lab, using Agrobacterium tumefaciens to inoculate pine and spruce somatic embryogenic cultures, was published (Wenck et al., 1999). In the work described in this publication, stable transformation of both species was achieved, but while plants were regenerated from the transformed spruce cultures, no plants could be obtained from the loblolly pine cultures.
U.S. Pat. No. 5,565,347 (Fillatti and Thomas, 1996) claims transformation of plants by co-cultivation of cotyledon shoot cultures with Agrobacterium, but again no example of transformation of any gymnosperm is given. Recovery of plants transformed via Agrobacterium from species of the subgenus Pinus via methods similar to those claimed in U.S. Pat. No. 5,565,347 has not been achieved with high frequency. There is a report of stable transformation of Pinus taeda specifically by inoculating shoot apices using the methods of U.S. Pat. No. 5,164,310 (Smith et al., 1992; which claimed the application of these methods to flowering plants, not conifers), but regeneration of transformed plants was a very low frequency occurrence. Stable transformation of Pinus radiata by inoculating cotyledons and later lateral buds has been reported publicly (Connett et al. 1993), but again regeneration of transformed plants was a very low frequency occurrence. Methods using shoot apices, lateral buds, cotyledons and similar tissue have a high probability of regenerating chimaeric plants. This, combined with the low frequency of regeneration, results in such methods being considered inviable for large-scale production of transformed plants.
Transformation of embryogenic cultures of gymnosperms has been a means of producing largely non-chimaeric transformed plants. Most reports of transformation of embryogenic cultures of gymnosperms, and all reports which featured regeneration of plants suitable for field planting from embryogenic cultures of pines of the subgenus Pinus, use biolistic transformation methods. However, those skilled in the art recognize that biolistic transformation methods have disadvantages relative to Agrobacterium-mediated transformation, such as the delivery of relatively smaller pieces of heterologous DNA in relatively higher copy numbers, with relatively more rearrangements seen on incorporation into the plant chromosomes.
Stable transformation of embryogenic cultures of Pinus strobus by Agrobacterium, followed by regeneration of plants, has been presented in a public forum (Séguin et al. 1999, IUFRO Wood Biotechnology conference). Pinus strobus is in the subgenus Strobus, or soft pines, while the Southern yellow pines such as Pinus taeda, Pinus elliotii, and Pinus caribaea, as well as the Eastern hard pines such as P. rigida, P. serotina, P. nigra and P. sylvestris, and the Western hard pines such as P. radiata and P. attenuata are in the subgenus Pinus. It is well known to those skilled in the art that the somatic embryogenesis systems for soft pines are different from those of the genetically different hard pines. Regeneration of plants following stable transformation of embryogenic cultures of any pine of the subgenus Pinus by Agrobacterium has not been reported in the literature.
A second problem, particularly relating to Agrobacterium transformation, has to do with the means of eradication of Agrobacterium following co-cultivation. Methods that have been employed in Agrobacterium transformation by those skilled in the art comprise physical washing of the bacteria from the plant cells and application of eradicants such as antibiotics in the plant culture media. Washing procedures are considered by those skilled in the art to be disadvantageous because they can result in significant loss of potentially transformed plant cells and damage to those that remain due to anaerobicity in the wash liquid, incomplete transfer, and shearing during movement of the cells from one medium to another. On the other hand certain eradicants, commonly used by those skilled in the art of transformation in order to kill the Agrobacterium, when incorporated into wash media or into media used for post-transformation recovery, selection, and/or proliferative growth, are detrimental to the subsequent differentiation of pine embryos that could give rise to transformed pine plants. In addition, eradicants incorporated into embryo development and maturation media are sometimes rendered partially or wholly inactive due to the high temperature of polymerization of the media. Moreover, the continuous incorporation of these eradicants in culture media is relatively expensive.
A third problem, relevant to any transformation method useful for groups of smaller, less differentiated cells such as precotyledonary somatic embryos, cell suspensions, or clumps of callus, is the detrimental nature of practices commonly used for post-transformation selection of transformed cells, which include laying the cells on filter papers or directly on the surface of gelled media. Detrimental conditions that can develop at the interfaces, such as anaerobicity, accumulation of exudates from necrotic cells, and barriers to diffusion of selection agents, nutrients, and plant growth regulators, are often exacerbated by incomplete transfer of cells from one medium to another, or transfer of cells with bits of spent media clinging to the desired material that also form a barrier to diffusion.
Thus, it is an object of the present invention to provide improved methods for the transformation of coniferous plants and the regeneration of transformed coniferous plants. These methods include improved methods for minimizing physical damage to cells during transformation and subsequent steps, for eradicating Agrobacterium from cell culture, for selecting genetically transformed pine cells, for growing pine cell cultures on “double layer” or “biphasic” culture systems, for transferring pine cell cultures between liquid and gelled media, gelled and liquid media, different liquid media or different gelled media, and for enhancing efficiency of regeneration with the use of certain components in the media.